Nucleic acid binding polypeptide library

ABSTRACT

The invention relates to a zinc finger polypeptide library in which each polypeptide comprises more than one zinc finger which has been at least partially randomized, and to a set of zinc finger polypeptide libraries which encode overlapping zinc finger polypeptides, each polypeptide comprising more than one zinc finger which has been at least partially randomized, and which polypeptide may be assembled after selection to form a multifinger zinc finger polypeptide.

The present invention relates to a library system for the selection ofzinc finger polypeptides. In particular, the invention relates to abinary system, in which zinc finger motifs are randomised in overlappingregions and to smart libraries incorporating limited directedrandomisation at selected positions.

Protein-nucleic acid recognition is a commonplace phenomenon which iscentral to a large number of biomolecular control mechanisms whichregulate the functioning of eukaryotic and prokaryotic cells. Forinstance, protein-DNA interactions form the basis of the regulation ofgene expression and are thus one of the subjects most widely studied bymolecular biologists.

A wealth of biochemical and structural information explains the detailsof protein-DNA recognition in numerous instances, to the extent thatgeneral principles of recognition have emerged. Many DNA-bindingproteins contain independently folded domains for the recognition ofDNA, and these domains in turn belong to a large number of structuralfamilies, such as the leucine zipper, the “helix-turn-helix” and zincfinger families.

Despite the great variety of structural domains, the specificity of theinteractions observed to date between protein and DNA most often derivesfrom the complementarity of the surfaces of a protein α-helix and themajor groove of DNA [Klug, (1993) Gene 135:83-92]. In light of therecurring physical interaction of α-helix and major groove, thetantalising possibility arises that the contacts between particularamino acids and DNA bases could be described by a simple set of rules;in effect a stereochemical recognition code which relates proteinprimary structure to binding-site sequence preference.

It is clear, however, that no code will be found which can describe DNArecognition by all DNA-binding proteins. The structures of numerouscomplexes show significant differences in the way that the recognitionα-helices of DNA-binding proteins from different structural familiesinteract with the major groove of DNA, thus precluding similarities inpatterns of recognition. The majority of known DNA-binding motifs arenot particularly versatile, and any codes which might emerge wouldlikely describe binding to a very few related DNA sequences.

Even within each family of DNA-binding proteins, moreover, it hashitherto appeared that the deciphering of a code would be elusive. Dueto the complexity of the protein-DNA interaction, there does not appearto be a simple “alphabetic” equivalence between the primary structuresof protein and nucleic acid which specifies a direct amino acid to baserelationship.

International patent application WO 96/06166 addresses this issue andprovides a “syllabic” code which explains protein-DNA interactions forzinc finger nucleic acid binding proteins. A syllabic code is a codewhich relies on more than one feature of the binding protein to specifybinding to a particular base, the features being combinable in the formsof “syllables”, or complex instructions, to define each specificcontact.

However, this code is incomplete, providing no specific instructionspermitting the specific selection of nucleotides other than G in the 5′position of each triplet. The method relies on randomisation andsubsequent selection in order to generate nucleic acid binding proteinsfor other specificities. Even with the aid of partial randomisation andselection, however, neither the method reported in WO 96/06166 nor anyother methods of the prior art have succeeded in isolating a zinc fingerpolypeptide based on the first finger of Zif268 capable of bindingtriplets wherein the 5′ base is other than G or T. This is a seriousshortfall in any ability to design zinc finger proteins.

Moreover, this document relies upon the notion that zinc fingers bind toa nucleic acid triplet or multiples thereof, as does all of the priorart. We have now determined that zinc finger binding sites aredetermined by overlapping 4 bp subsites, and that sequence-specificityat the boundary between subsites arises from synergy between adjacentfingers. This has important implications for the design and selection ofzinc fingers with novel DNA binding specificities.

SUMMARY OF THE INVENTION

The present invention recognises the importance of overlapping 4 bpsubsite recognition in zinc finger polypeptide design. The resultantsynergy between zinc fingers is overlooked in classical zinc fingerlibrary design, in which only a single zinc finger is randomised in eachlibrary.

Accordingly, the present invention provides a zinc finger polypeptidelibrary in which each polypeptide comprises more than one zinc fingerwhich has been at least partially randomised.

Preferably, the invention provides a group of zinc finger polypeptidelibraries which encode overlapping zinc finger polypeptides, eachpolypeptide comprising more than one zinc finger which has been at leastpartially randomised, and which polypeptides may be assembled afterselection to form a multifinger zinc finger polypeptide.

In a further aspect, the invention relates to a library as describedabove in which randomisation is limited to substituting amino acidswhich are known to dictate variation in binding site specificity. Thepresent invention provides a code of amino acid position bias whichpermits the selection of the library against any nucleic acid sequenceas the target sequence, and the production of a specific nucleicacid-binding protein which will bind thereto. Moreover, the inventionprovides a method by which a zinc finger protein specific for any givennucleic acid sequence may be designed and optimised. The presentinvention therefore concerns a recognition bias which has beenelucidated for the interactions of classical zinc fingers with nucleicacid. In this case a pattern of rules is provided which covers bindingto all nucleic acid sequences.

The code set forth in the present invention takes account of synergisticinteractions between adjacent zinc fingers, thereby allowing theselection of any desired binding site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates zinc finger-DNA interactions. A: model of classicaltriplet interactions with DNA base triplets in Zif268; B: similar modelshowing quadruplet interactions; C: model of library design forrecognition code determination.

FIG. 2 shows the amino acid sequence of three fingers used for phagedisplay selection in the determination of recognition code.

FIG. 3 lists the sequence-specific zinc finger clones obtained fromphage selections, and their binding site signatures.

FIG. 4 shows the base/amino acid correlation of the clones isolated fromphage selections. Recognition patterns are highlighted.

FIG. 5 illustrates the sequence-specific interactions selected for atposition 2 of the α-helix, binding to position 1 of the quadruplet.

FIG. 6 is a schematic diagram of the construction of a library accordingto the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to libraries. The term “library” is usedaccording to its common usage in the art, to denote a collection ofpolypeptides or, preferably, nucleic acids encoding polypeptides. Thepolypeptides of the invention contain regions of randomisation, suchthat each library will comprise or encode a repertoire of polypeptides,wherein individual polypeptides differ in sequence from each other. Thesame principle is present in virtually all libraries developed forselection, such as by phage display.

Randomisation, as used herein, refers to the variation of the sequenceof the polypeptides which comprise the library, such that various aminoacids may be present at any given position in different polypeptides.Randomisation may be complete, such that any amino acid may be presentat a given position, or partial, such that only certain amino acids arepresent. Preferably, the randomisation is achieved by mutagenesis at thenucleic acid level, for example by synthesising novel genes encodingmutant proteins and expressing these to obtain a variety of differentproteins. Alternatively, existing genes can be themselves mutated, suchby site-directed or random mutagenesis, in order to obtain the desiredmutant genes.

Mutations may be performed by any method known to those of skill in theart. Preferred, however, is site-directed mutagenesis of a nucleic acidsequence encoding the protein of interest. A number of methods forsite-directed mutagenesis are known in the art, from methods employingsingle-stranded phage such as M13 to PCR-based techniques (see “PCRProtocols: A guide to methods and applications”, M. A. Innis, D. H.Gelfand, J. J. Sninsky, T. J. White (eds.). Academic Press, New York,1990). Preferably, the commercially available Altered Site IIMutagenesis System (Promega) may be employed, according to thedirections given by the manufacturer.

Screening of the proteins produced by mutant genes is preferablyperformed by expressing the genes and assaying the binding ability ofthe protein product. A simple and advantageously rapid method by whichthis may be accomplished is by phage display, in which the mutantpolypeptides are expressed as fusion proteins with the coat proteins offilamentous bacteriophage, such as the minor coat protein pII ofbacteriophage m13 or gene III of bacteriophage Fd, and displayed on thecapsid of bacteriophage transformed with the mutant genes. The targetnucleic acid sequence is used as a probe to bind directly to the proteinon the phage surface and select the phage possessing advantageousmutants, by affinity purification. The phage are then amplified bypassage through a bacterial host, and subjected to further rounds ofselection and amplification in order to enrich the mutant pool for thedesired phage and eventually isolate the preferred clone(s). Detailedmethodology for phage display is known in the art and set forth, forexample, in U.S. Pat. No. 5,223,409; Choo and Klug, (1995) CurrentOpinions in Biotechnology 6:431-436; Smith, (1985) Science228:1315-1317; and McCafferty et al., (1990) Nature 348:552-554; allincorporated herein by reference. Vector systems and kits for phagedisplay are available commercially, for example from Pharmacia.

The polypeptides which comprise the libraries according to the inventionare zinc finger polypeptides. In other words, they comprise a Cys2-His2zinc finger motif. It is a feature of the invention that eachpolypeptide comprises more then one zinc finger, such that the librarymay be selected on the basis of the interaction between two or more zincfingers on the polypeptide.

Zinc fingers, as is known in the art, are nucleic acid bindingmolecules. Each zinc finger binds to a quadruplet sequence in a targetnucleic acid through contacts between specific amino acid residues ofthe α-helix of the zinc finger and the nucleic acid strand. Thequadruplets specified in the present invention are overlapping, suchthat, when read 3′ to 5′ on the − strand of the nucleic acid, base 4 ofthe first quadruplet is base 1 of the second, and so on. Accordingly, inthe present application, the bases of each quadruplet are referred bynumber, from 1 to 4, 1 being the 3′ base and 4 being the 5′ base. Base 4is equivalent to the 5′ base of a classical zinc finger binding triplet.In general, base 4 is bound through a contact at position +6 of theα-helix, base 3 through a contact at position +3, base 2 through acontact at position −1 and base 1 through a contact to the oppositestrand of double-stranded nucleic acids at position +2.

All of the nucleic acid-binding residue positions of zinc fingers, asreferred to herein, are numbered from the first residue in the α-helixof the finger, ranging from +1 to +9. “−1” refers to the residue in theframework structure immediately preceding the α-helix in a Cys2-His2zinc finger polypeptide.

Residues referred to as “++2” are residues present in an adjacent(C-terminal) finger. They reflect the synergistic cooperation betweenposition +2 on base 1 (on the + strand) and position +6 of the preceding(N-terminal) finger on base 4 of the preceding (3′) quadruplet, which isthe same base due to the overlap. Where there is no C-terminal adjacentfinger, “++” interactions do not operate.

Cys2-His2 zinc finger binding proteins, as is well known in the art,bind to target nucleic acid sequences via α-helical zinc metal atomco-ordinated binding motifs known as zinc fingers. Each zinc finger in azinc finger nucleic acid binding protein is responsible for determiningbinding to a nucleic acid quadruplet in a nucleic acid binding sequence.Preferably, there are 2 or more zinc fingers, for example 2, 3, 4, 5 or6 zinc fingers, in each binding protein. Advantageously, there are 3zinc fingers in each zinc finger binding protein.

The present invention allows the production of what are essentiallyartificial nucleic acid binding proteins. In these proteins, artificialanalogues of amino acids may be used, to impart the proteins withdesired properties or for other reasons. Thus, the term “amino acid”,particularly in the context where “any amino acid” is referred to, meansany sort of natural or artificial amino acid or amino acid analogue thatmay be employed in protein construction according to methods known inthe art. Moreover, any specific amino acid referred to herein may bereplaced by a functional analogue thereof, particularly an artificialfunctional analogue. The nomenclature used herein therefore specificallycomprises within its scope functional analogues of the defined aminoacids.

The α-helix of a zinc finger binding protein aligns antiparallel to thenucleic acid strand, such that the primary nucleic acid sequence isarranged 3′ to 5′ in order to correspond with the N terminal toC-terminal sequence of the zinc finger. Since nucleic acid sequences areconventionally written 5′ to 3′, and amino acid sequences N-terminus toC-terminus, the result is that when a nucleic acid sequence and a zincfinger protein are aligned according to convention, the primaryinteraction of the zinc finger is with the − strand of the nucleic acid,since it is this strand which is aligned 3′ to 5′. These conventions arefollowed in the nomenclature used herein. It should be noted, however,that in nature certain fingers, such as finger 4 of the protein GLI,bind to the + strand of nucleic acid: see Suzuki et al., (1994) NAR22:3397-3405 and Pavletich and Pabo, (1993) Science 261:1701-1707. Theincorporation of such fingers into nucleic acid binding moleculesaccording to the invention is envisaged.

The libraries of the present invention allow selection for synergisticcooperation between adjacent zinc fingers by promoting coselection ofadjacent fingers against a single DNA target. This is achieved byrandomising, in the same zinc finger polypeptide, more than one zincfinger. In a preferred embodiment, approximately one and a half zincfingers are randomised in each polypeptide, but this may be variedaccording to library design.

The zinc finger polypeptides encoded in the library of the invention maycomprise any number of zinc fingers, provided this is more than one.Advantageously, each polypeptide encodes between three and six zincfingers. In each library, the randomisation extends to cover the overlapof at least one pair of zinc fingers. Preferably, the overlap of asingle pair is covered.

Preferably, the libraries of the present invention are provided as sets.Thus, a three zinc finger polypeptide comprising fingers F1, F2 and F3may be presented in a set of two libraries, each library comprising atwo zinc finger polypeptide. A first library is composed of polypeptidesconsisting essentially of F1 and F2, whilst a second library is composedof polypeptides consisting essentially of F2 and F3. The randomisationin each library includes the overlap between F1 and F2, and F2 and F3respectively.

Preferably, each library will comprise randomisation at at leastposition 6 of a first finger and position 2 of a second finger. Sincethese residues contact the same base pair on a double stranded nucleicacid target, it is advantageous that they be varied together.

In the case of a three zinc finger polypeptide, the first library willbe randomised in fingers F1 and F2, whilst the second is randomised inF2 and F3. Polypeptides may be recombined, post-selection, in the F2sequence to create a single polypeptide containing F1, F2 and F3. Thispolypeptide will have been selected taking into account the overlapbetween F1 and F2, and F2 and F3.

Advantageously, a greater number of position may be varied in each zincfinger. Preferably, residues selected from positions −1, 1, 2, 3 5 and 6are varied in a first zinc finger and positions −1, 1, 2 and 3 in asecond. In a companion library, positions 3, 5 and 6 may be varied inthe second finger, and positions −1, 1, 2 and 3 in a third finger. Inthe final finger (in the case of a three finger protein this will be thethird finger), residues 5 and 6 may also be varied.

In order that the libraries may be recombined after selection, thepolypeptides are preferably designed to include a suitable restrictionsite in the nucleic acid encoding the zinc finger shared by twolibraries. The position of the cleavage site will dictate the precisesite of the variations made in the shared zinc finger in each library.Thus, in a set of two libraries encoding a three zinc finger protein, ifthe cleavage site is between positions 3 and 5 of the α-helix, positions3 and 5 may be randomised in a first library and positions 5 and 6 in asecond.

Although it is preferred that residues for randomisation or variation beselected from positions −1, 1, 2, 3, 5 and 6, further residues may alsobe randomised. For example, the randomisation of position 8 may beadvantageous. Moreover, it is envisaged that fewer than all of the givenpositions are randomised.

In a preferred embodiment, a two-library system for selection of athree-finger protein is varied at F1 positions −1, 2, 3 5, and 6 and F2positions −1, 1, 2 and 3 in the first library. The second library isvaried at F2 positions 3 and 6 and F3 positions −1, 1, 2, 3, 5 and 6. Inthis case, the cleavage and recombination point will be between residues3 and 5, preferably between residues 4 and 5, of the α-helix of F2.

Subsequent to the recombination event, recombined polypeptide-encodingnucleic acids may be expressed in suitable expression systems, or clonedinto Fd phage for further selection.

In a preferred aspect of the present invention, the libraries of theinvention are not truly randomised at the selected positions, but onlypartially randomised so that certain but not all amino acids areencoded. This strategy may be used for two purposes.

In a first embodiment, variation is restricted to those amino acidswhich are known to be capable of directing sequence-specific binding ofnucleic acid target sequences when incorporated at a given position inthe α-helix of a zinc finger. It is known that certain amino acids arenot suitable for incorporation at certain positions, irrespective oftarget sequence. These amino acids are avoided.

In a second embodiment, variation is restricted to those amino acidswhich are known to be capable of directing sequence-specific binding ofnucleic acid target sequences when incorporated at a given position inthe ax-helix of a zinc finger, and variation is directed to specifythose residues which are known to favour binding to a specific targetsequence at any given position. Thus, the invention permits the designof dedicated libraries from which polypeptides capable of binding tospecific target sequence, or to a series of related target sequences,may be selected.

In the first embodiment, which provides a library system for generalapplication, randomisation is preferably effected at all of thepositions indicated above. Preferably, the amino acids selected toappear at each given position are as set forth in Table 1: TABLE 1Position Possible Amino Acids −1 R, Q, H, N, D, A, T 1 S, R, K, N 2 D,A, R, Q, H, K, S, N 3 H, N, S, T, V, A, D 5 I, T, K 6 R, Q, V, A, E, K,N, T

It is not necessary for each finger to be randomised at each of thepositions given in table 1. In a preferred embodiment, a library forselecting a three-finger protein is constructed according to thespecifications given in Table 2: TABLE 2 Library 1 Library 2 amino acidamino acid F1: F1: −1 R, Q, H, N, D, A 2 D, A, R, Q, H, K, S, N 3 H, N,S, T, V, A, D 5 I, T 6 R, Q, V, A, E, K, N, T F2 −1 R, Q, H, N, D, A, T1 S, R 2 D, A, R, Q, H, K, S, N 3 H, N, S, T, V, A, D 3 H, N, S, T, V,A, D 6 R, Q, V, A, E, K, N, T F3 −1 R, Q, H, N, D, A, T 1 R, K, S, N 2D, A, R, Q, H, K, S, N 3 H, N, S, T, V, A, D 5 K, I, T 6 R, Q, V, A, E,K, N, T

In the second embodiment, the identity of each amino acid at anyparticular position is selected according to zinc finger recognitionrules as provided herein. In a preferred aspect, therefore, theinvention provides a method for preparing a nucleic acid binding proteinof the Cys2-His2 zinc finger class capable of binding to a nucleic acidquadruplet in a target nucleic acid sequence, wherein binding to eachbase of the quadruplet by an α-helical zinc finger nucleic acid bindingmotif in the protein is determined as follows:

a) if base 4 in the quadruplet is G, then position +6 in the α-helix isArg or Lys;

b) if base 4 in the quadruplet is A, then position +6 in the α-helix isGlu, Asn or Val;

c) if base 4 in the quadruplet is T, then position +6 in the α-helix isSer, Thr, Val or Lys;

d) if base 4 in the quadruplet is C, then position +6 in the α-helix isSer, Thr, Val, Ala, Glu or Asn;

e) if base 3 in the quadruplet is G, then position +3 in the α-helix isHis;

f) if base 3 in the quadruplet is A; then position +3 in the α-helix isAsn;

g) if base 3 in the quadruplet is T, then position +3 in the α-helix isAla, Ser or Val; provided that if it is Ala, then one of the residues at−1 or +6 is a small residue;

h) if base 3 in the quadruplet is C, then position +3 in the α-helix isSer, Asp, Glu, Leu, Thr or Val;

i) if base 2 in the quadruplet is G, then position −1 in the α-helix isArg;

j) if base 2 in the quadruplet is A, then position −1 in the α-helix isGln;

k) if base 2 in the quadruplet is T, then position −1 in the α-helix isHis or Thr;

l) if base 2 in the quadruplet is C, then position −1 in the α-helix isAsp or His.

m) if base 1 in the quadruplet is G, then position +2 is Glu;

n) if base 1 in the quadruplet is A, then position +2 Arg or Gln;

o) if base 1 in the quadruplet is C, then position +2 is Asn, Gln, Arg,His or Lys;

p) if base 1 in the quadruplet is T, then position +2 is Ser or Thr.

The foregoing represents a set of rules which permits the design of azinc finger binding protein specific for any given nucleic acidsequence. A novel finding related thereto is that position +2 in thehelix is responsible for determining the binding to base 1 of thequadruplet. In doing so, it cooperates synergistically with position +6,which determines binding at base 4 in the quadruplet, bases 1 and 4being overlapping in adjacent quadruplets.

Although zinc finger polypeptides are considered to bind to overlappingquadruplet sequences, the method of the present invention allowspolypeptides to be designed to bind to target sequences which are notmultiples of overlapping quadruplets. For example, a zinc fingerpolypeptide may be designed to bind to a palindromic target sequence.Such sequences are commonly found as, for example, restriction enzymetarget sequences.

Preferably, creation of zinc fingers which bind to fewer than threenucleotides is achieved by specifying, in the zinc finger, amino acidswhich are unable to support H-bonding with the nucleic acid in therelevant position.

Advantageously, this is achieved by substituting Gly at position −1 (toeliminate a contact with base 2) and/or Ala at positions +3 and/or +6(to eliminate contacts at the 3rd or 4th base respectively).

Preferably, the contact with the final (3′) base in the target sequenceshould be strengthened, if necessary, by substituting a residue at therelevant position which is capable of making a direct contact with thephosphate backbone of the nucleic acid.

These and other considerations may be incorporated in a library set inaccordance with the invention.

A zinc finger binding motif is a structure well known to those in theart and defined in, for example, Miller et al., (1985) EMBO J.4:1609-1614; Berg (1988) PNAS (USA) 85:99-102; Lee et al., (1989)Science 245:635-637; see International patent applications WO 96/06166and WO 96/32475, corresponding to U.S. Ser. No. 081422,107, incorporatedherein by reference.

As used herein, “nucleic acid” refers to both RNA and DNA, constructedfrom natural nucleic acid bases or synthetic bases, or mixtures thereof.Preferably, however, the binding proteins of the invention are DNAbinding proteins.

In general, a preferred zinc finger framework has the structure:X₀₋₂ C X₁₋₅ C X₉₋₁₄ H X₃₋₆ ^(H)/_(C)   (A)

where X is any amino acid, and the numbers in subscript indicate thepossible numbers of residues represented by X.

In a preferred aspect of the present invention, zinc finger nucleic acidbinding motifs may be represented as motifs having the following primarystructure:X^(a) C X₂₋₄ C X₂₋₃ F X^(c) X X X X L X X H X X X^(b) H—linker −1 1 2 34 5 6 7 8 9   (B)

wherein X (including X^(a), X^(b) and X^(c)) is any amino acid. X₂₋₄ andX₂₋₃ refer to the presence of 2 or 4, or 2 or 3, amino acids,respectively. The Cys and His residues, which together co-ordinate thezinc metal atom, are marked in bold text and are usually invariant, asis the Leu residue at position +4 in the α-helix.

Modifications to this representation may occur or be effected withoutnecessarily abolishing zinc finger function, by insertion, mutation ordeletion of amino acids. For example it is known that the second Hisresidue may be replaced by Cys (Krizek et al., (1991) J. Am. Chem. Soc.113:4518-4523) and that Leu at +4 can in some circumstances be replacedwith Arg. The Phe residue before X_(c) may be replaced by any aromaticother than Trp. Moreover, experiments have shown that departure from thepreferred structure and residue assignments for the zinc finger aretolerated and may even prove beneficial in binding to certain nucleicacid sequences. Even taking this into account, however, the generalstructure involving an α-helix co-ordinated by a zinc atom whichcontacts four Cys or His residues, does not alter. As used herein,structures (A) and (B) above are taken as an exemplary structurerepresenting all zinc finger structures of the Cys2-His2 type.

Preferably, X^(a) is ^(F)/_(Y)-X or P-^(F)/_(Y)-X. In this context, X isany amino acid. Preferably, in this context X is E, K, T or S. Lesspreferred but also envisaged are Q, V, A and P. The remaining aminoacids remain possible.

Preferably, X₂₋₄ consists of two amino acids rather than four. The firstof these amino acids may be any amino acid, but S, E, K, T, P and R arepreferred. Advantageously, it is P or R. The second of these amino acidsis preferably E, although any amino acid may be used.

Preferably, X^(b) is T or I.

Preferably, X^(c) is S or T.

Preferably, X₂₋₃ is G-K-A, G-K-C, G-K-S or G-K-G. However, departuresfrom the preferred residues are possible, for example in the form ofM-R-N or M-R.

Preferably, the linker is T-G-E-K or T-G-E-K-P.

As set out above, the major binding interactions occur with amino acids−1, +2, +3 and +6. Amino acids +4 and +7 are largely invariant. Theremaining amino acids may be essentially any amino acids. Preferably,position +9 is occupied by Arg or Lys. Advantageously, positions +1, +5and +8 are not hydrophobic amino acids, that is to say are not Phe, Trpor Tyr.

In a most preferred aspect, therefore, bringing together the above, theinvention allows the definition of every residue in a zinc fingernucleic acid binding motif which will bind specifically to a givennucleic acid quadruplet.

The code provided by the present invention is not entirely rigid;certain choices are provided. For example, positions +1, +5 and +8 mayhave any amino acid allocation, whilst other positions may have certainoptions: for example, the present rules provide that, for binding to acentral T residue, any one of Ala, Ser or Val may be used at +3. In itsbroadest sense, therefore, the present invention provides a very largenumber of proteins which are capable of binding to every defined targetnucleic acid quadruplet.

Preferably, however, the number of possibilities may be significantlyreduced. For example, the non-critical residues +1, +5 and +8 may beoccupied by the residues Lys, Thr and Gln respectively as a defaultoption. In the case of the other choices, for example, the first-givenoption may be employed as a default. Thus, the code according to thepresent invention allows the design of a single, defined polypeptide (a“default” polypeptide) which will bind to its target quadruplet.

In a further aspect of the present invention, there is provided a methodfor preparing a nucleic acid binding protein of the Cys2-His2 zincfinger class capable of binding to a target nucleic acid sequence,comprising the steps of:

a) selecting a model zinc finger domain from the group consisting ofnaturally occurring zinc fingers and consensus zinc fingers; and

b) mutating one or more of positions −1, +2, +3 and +6 of the finger asrequired according to the rules set forth above.

In general, naturally occurring zinc fingers may be selected from thosefingers for which the nucleic acid binding specificity is known. Forexample, these may be the fingers for which a crystal structure has beenresolved: namely Zif 268 (Elrod-Erickson et al., (1996) Structure4:1171-1180), GLI (Pavletich and Pabo, (1993) Science 261:1701-1707),Tramtrack (Fairall et al., (1993) Nature 366:483-487) and YY1 (Houbaviyet al., (1996) PNAS (USA) 93:13577-13582).

The naturally occurring zinc finger 2 in Zif 268 makes an excellentstarting point from which to engineer a zinc finger and is preferred.

Consensus zinc finger structures may be prepared by comparing thesequences of known zinc fingers, irrespective of whether their bindingdomain is known. Preferably, the consensus structure is selected fromthe group consisting of the consensus structure P Y K C P E C G K S F SQ K S D L V K H Q R T H T G, and the consensus structure P Y K C S E C GK A F S Q K S N L T R H Q R I H T G E K P.

The consensuses are derived from the consensus provided by Krizek etal., (1991) J. Am. Chem. Soc. 113:4518-4523 and from Jacobs, (1993) PhDthesis, University of Cambridge, UK. In both cases, the linker sequencesdescribed above for joining two zinc finger motifs together, namely TGEKor TGEKP can be formed on the ends of the consensus. Thus, a P may beremoved where necessary, or, in the case of the consensus terminating TG, E K (P) can be added.

When the nucleic acid specificity of the model finger selected is known,the mutation of the finger in order to modify its specificity to bind tothe target nucleic acid may be directed to residues known to affectbinding to bases at which the natural and desired targets differ.Otherwise, mutation of the model fingers should be concentrated uponresidues −1, +2, +3 and +6 as provided for in the foregoing rules.

In order to produce a binding protein having improved binding, moreover,the rules provided by the present invention may be supplemented byphysical or virtual modelling of the protein/nucleic acid interface inorder to assist in residue selection.

Zinc finger binding motifs designed according to the invention may becombined into nucleic acid binding proteins having a multiplicity ofzinc fingers. Preferably, the proteins have at least two zinc fingers.In nature, zinc finger binding proteins commonly have at least threezinc fingers, although two-zinc finger proteins such as Tramtrack areknown. The presence of at least three zinc fingers is preferred. Bindingproteins may be constructed by joining the required fingers end to end,N-terminus to C-terminus. Preferably, this is effected by joiningtogether the relevant nucleic acid coding sequences encoding the zincfingers to produce a composite coding sequence encoding the entirebinding protein. The invention therefore provides a method for producinga nucleic acid binding protein as defined above, wherein the nucleicacid binding protein is constructed by recombinant DNA technology, themethod comprising the steps of:

a) preparing a nucleic acid coding sequence encoding two or more zincfinger binding motifs as defined above, placed N-terminus to C-terminus;

b) inserting the nucleic acid sequence into a suitable expressionvector; and

c) expressing the nucleic acid sequence in a host organism in order toobtain the nucleic acid binding protein.

A “leader” peptide may be added to the N-terminal finger. Preferably,the leader peptide is MAEEKP.

The nucleic acid encoding the nucleic acid binding protein according tothe invention can be incorporated into vectors for further manipulation.As used herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous nucleic acid into cells for eitherexpression or replication thereof. Selection and use of such vehiclesare well within the skill of the person of ordinary skill in the art.Many vectors are available, and selection of appropriate vector willdepend on the intended use of the vector, i.e. whether it is to be usedfor DNA amplification or for nucleic acid expression, the size of theDNA to be inserted into the vector, and the host cell to be transformedwith the vector. Each vector contains various components depending onits function (amplification of DNA or expression of DNA) and the hostcell for which it is compatible. The vector components generallyinclude, but are not limited to, one or more of the following: an originof replication, one or more marker genes, an enhancer element, apromoter, a transcription termination sequence and a signal sequence.

Both expression and cloning vectors generally contain nucleic acidsequence that enable the vector to replicate in one or more selectedhost cells. Typically in cloning vectors, this sequence is one thatenables the vector to replicate independently of the host chromosomalDNA, and includes origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2 μ plasmid origin issuitable for yeast, and various viral origins (e.g. SV 40, polyoma,adenovirus) are useful for cloning vectors in mammalian cells.Generally, the origin of replication component is not needed formammalian expression vectors unless these are used in mammalian cellscompetent for high level DNA replication, such as COS cells.

Most expression vectors are shuttle vectors, i.e. they are capable ofreplication in at least one class of organisms but can be transfectedinto another class of organisms for expression. For example, a vector iscloned in E. coli and then the same vector is transfected into yeast ormammalian cells even though it is not capable of replicatingindependently of the host cell chromosome. DNA may also be replicated byinsertion into the host genome. However, the recovery of genomic DNAencoding the nucleic acid binding protein is more complex than that ofexogenously replicated vector because restriction enzyme digestion isrequired to excise nucleic acid binding protein DNA. DNA can beamplified by PCR and be directly transfected into the host cells withoutany replication component.

Advantageously, an expression and cloning vector may contain a selectiongene also referred to as selectable marker. This gene encodes a proteinnecessary for the survival or growth of transformed host cells grown ina selective culture medium. Host cells not transformed with the vectorcontaining the selection gene will not survive in the culture medium.Typical selection genes encode proteins that confer resistance toantibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate ortetracycline, complement auxotrophic deficiencies, or supply criticalnutrients not available from complex media.

As to a selective gene marker appropriate for yeast, any marker gene canbe used which facilitates the selection for transformants due to thephenotypic expression of the marker gene. Suitable markers for yeastare, for example, those conferring resistance to antibiotics G418,hygromycin or bleomycin, or provide for prototrophy in an auxotrophicyeast mutant, for example the URA3, LEU2, LYS2, TRP1, or HIS3 gene.

Since the replication of vectors is conveniently done in E. coli, an E.coli genetic marker and an E. coli origin of replication areadvantageously included. These can be obtained from E. coli plasmids,such as pBR322, Bluescript© vector or a pUC plasmid, e.g. pUC18 orpUC19, which contain both E. coli replication origin and E. coli geneticmarker conferring resistance to antibiotics, such as ampicillin.

Suitable selectable markers for mammalian cells are those that enablethe identification of cells competent to take up nucleic acid bindingprotein nucleic acid, such as dihydrofolate reductase (DHFR,methotrexate resistance), thymidine kinase, or genes conferringresistance to G418 or hygromycin. The mammalian cell transformants areplaced under selection pressure which only those transformants whichhave taken up and are expressing the marker are uniquely adapted tosurvive. In the case of a DHFR or glutamine synthase (GS) marker,selection pressure can be imposed by culturing the transformants underconditions in which the pressure is progressively increased, therebyleading to amplification (at its chromosomal integration site) of boththe selection gene and the linked DNA that encodes the nucleic acidbinding protein. Amplification is the process by which genes in greaterdemand for the production of a protein critical for growth, togetherwith closely associated genes which may encode a desired protein, arereiterated in tandem within the chromosomes of recombinant cells.Increased quantities of desired protein are usually synthesised fromthus amplified DNA.

Expression and cloning vectors usually contain a promoter that isrecognised by the host organism and is operably linked to nucleic acidbinding protein encoding nucleic acid. Such a promoter may be inducibleor constitutive. The promoters are operably linked to DNA encoding thenucleic acid binding protein by removing the promoter from the sourceDNA by restriction enzyme digestion and inserting the isolated promotersequence into the vector. Both the native nucleic acid binding proteinpromoter sequence and many heterologous promoters may be used to directamplification and/or expression of nucleic acid binding protein encodingDNA.

Promoters suitable for use with prokaryotic hosts include, for example,the β-lactamase and lactose promoter systems, alkaline phosphatase, thetryptophan (Trp) promoter system and hybrid promoters such as the tacpromoter. Their nucleotide sequences have been published, therebyenabling the skilled worker operably to ligate them to DNA encodingnucleic acid binding protein, using linkers or adapters to supply anyrequired restriction sites. Promoters for use in bacterial systems willalso generally contain a Shine-Delgarno sequence operably linked to theDNA encoding the nucleic acid binding protein.

Preferred expression vectors are bacterial expression vectors whichcomprise a promoter of a bacteriophage such as phagex or T7 which iscapable of functioning in the bacteria. In one of the most widely usedexpression systems, the nucleic acid encoding the fusion protein may betranscribed from the vector by T7 RNA polymerase (Studier et al, Methodsin Enzymol. 185; 60-89, 1990). In the E. coli BL21(DE3) host strain,used in conjunction with pET vectors, the T7 RNA polymerase is producedfrom the λ-lysogen DE3 in the host bacterium, and its expression isunder the control of the IPTG inducible lac UV5 promoter. This systemhas been employed successfully for over-production of many proteins.Alternatively the polymerase gene may be introduced on a lambda phage byinfection with an int-phage such as the CE6 phage which is commerciallyavailable (Novagen, Madison, USA). other vectors include vectorscontaining the lambda PL promoter such as PLEX (Invitrogen, NL), vectorscontaining the trc promoters such as pTrcHisXpress™ (Invitrogen) orpTrc99 (Pharmacia Biotech, SE) or vectors containing the tac promotersuch as pKK223-3 (Pharmacia Biotech) or PMAL (New England Biolabs, MA,USA).

Moreover, the nucleic acid binding protein gene according to theinvention preferably includes a secretion sequence in order tofacilitate secretion of the polypeptide from bacterial hosts, such thatit will be produced as a soluble native peptide rather than in aninclusion body. The peptide may be recovered from the bacterialperiplasmic space, or the culture medium, as appropriate:

Suitable promoting sequences for use with yeast hosts may be regulatedor constitutive and are preferably derived from a highly expressed yeastgene, especially a Saccharomyces cerevisiae gene. Thus, the promoter ofthe TRP1 gene, the ADHI or ADHII gene, the acid phosphatase. (PH05)gene, a promoter of the yeast mating pheromone genes coding for the a-or α-factor or a promoter derived from a gene encoding a glycolyticenzyme such as the promoter of the enolase, glyceraldehyde-3-phosphatedehydrogenase (GAP), 3-phospho glycerate kinase (PGK), hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphateisomerase, phosphoglucose isomerase or glucokinase genes, or a promoterfrom the TATA binding protein (TBP) gene can be used. Furthermore, it ispossible to use hybrid promoters comprising upstream activationsequences (UAS) of one yeast gene and downstream promoter elementsincluding a functional TATA box of another yeast gene, for example ahybrid promoter including the UAS(s) of the yeast PH05 gene anddownstream promoter elements including a functional TATA box of theyeast GAP gene (PH05-GAP hybrid promoter). A suitable constitutive PHO5promoter is e.g. a shortened acid phosphatase PH05 promoter devoid ofthe upstream regulatory elements (UAS) such as the PH05 (−173) promoterelement starting at nucleotide −173 and ending at nucleotide −9 of thePH05 gene.

Nucleic acid binding protein gene transcription from vectors inmammalian hosts may be controlled by promoters derived from the genomesof viruses such as polyoma virus, adenovirus, fowlpox virus, bovinepapilloma virus, avian sarcoma virus, cytomegalovirus (CMV), aretrovirus and Simian Virus 40 (SV40), from heterologous mammalianpromoters such as the actin promoter or a very strong promoter, e.g. aribosomal protein promoter, and from the promoter normally associatedwith nucleic acid binding protein sequence, provided such promoters arecompatible with the host cell systems.

Transcription of a DNA encoding nucleic acid binding protein by highereukaryotes may be increased by inserting an enhancer sequence into thevector. Enhancers are relatively orientation and position independent.Many enhancer sequences are known from mammalian genes (e.g. elastaseand globin). However, typically one will employ an enhancer from aeukaryotic cell virus. Examples include the SV40 enhancer on the lateside of the replication origin (bp 100-270) and the CMV early promoterenhancer. The enhancer may be spliced into the vector at a position 5′or 3′ to nucleic acid binding protein DNA, but is preferably located ata site 5′ from the promoter.

Advantageously, a eukaryotic expression vector encoding a nucleic acidbinding protein according to the invention may comprise a locus controlregion (LCR). LCRs are capable of directing high-level integration siteindependent expression of transgenes integrated into host cellchromatin, which is of importance especially where the nucleic acidbinding protein gene is to be expressed in the context of apermanently-transfected eukaryotic cell line in which chromosomalintegration of the vector has occurred, or in transgenic animals.

Eukaryotic vectors may also contain sequences necessary for thetermination of transcription and for stabilising the mRNA. Suchsequences are commonly available from the 5′ and 3′ untranslated regionsof eukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding nucleic acid binding protein.

An expression vector includes any vector capable of expressing nucleicacid binding protein nucleic acids that are operatively linked withregulatory sequences, such as promoter regions, that are capable ofexpression of such DNAs. Thus, an expression vector refers to arecombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector, that upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those with ordinaryskill in the art and include those that are replicable in eukaryoticand/or prokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome. For example, DNAs encoding nucleicacid binding protein may be inserted into a vector suitable forexpression of cDNAs in mammalian cells, e.g. a CMV enhancer-based vectorsuch as pEVRF (Matthias, et al., (1989) NAR 17, 6418).

Particularly useful for practising the present invention are expressionvectors that provide for the transient expression of DNA encodingnucleic acid binding protein in mammalian cells. Transient expressionusually involves the use of an expression vector that is able toreplicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector, and, in turn,synthesises high levels of nucleic acid binding protein. For thepurposes of the present invention, transient expression systems areuseful e.g. for identifying nucleic acid binding protein mutants, toidentify potential phosphorylation sites, or to characterise functionaldomains of the protein.

Construction of vectors according to the invention employs conventionalligation techniques. Isolated plasmids or DNA fragments are cleaved,tailored, and religated in the form desired to generate the plasmidsrequired. If desired, analysis to confirm correct sequences in theconstructed plasmids is performed in a known fashion. Suitable methodsfor constructing expression vectors, preparing in vitro transcripts,introducing DNA into host cells, and performing analyses for assessingnucleic acid binding protein expression and function are known to thoseskilled in the art. Gene presence, amplification and/or expression maybe measured in a sample directly, for example, by conventional Southernblotting, Northern blotting to quantitate the transcription of mRNA, dotblotting (DNA or RNA analysis), or in situ hybridisation, using anappropriately labelled probe which may be based on a sequence providedherein. Those skilled in the art will readily envisage how these methodsmay be modified, if desired.

In accordance with another embodiment of the present invention, thereare provided cells containing the above-described nucleic acids. Suchhost cells such as prokaryote, yeast and higher eukaryote cells may beused for replicating DNA and producing the nucleic acid binding protein.Suitable prokaryotes include eubacteria, such as Gram-negative orGram-positive organisms, such as E. coli, e.g. E. coli K-12 strains,DH5a and HB101. or Bacilli. Further hosts suitable for the nucleic acidbinding protein encoding vectors include eukaryotic microbes such asfilamentous fungi or yeast, e.g. Saccharomyces cerevisiae. Highereukaryotic cells include insect and vertebrate cells, particularlymammalian cells including human cells or nucleated cells from othermulticellular organisms. In recent years propagation of vertebrate cellsin culture (tissue culture) has become a routine procedure. Examples ofuseful mammalian host cell lines are epithelial or fibroblastic celllines such as Chinese hamster ovary (CHO) cells, NIH 3T3 cells, HeLacells or 293T cells. The host cells referred to in this disclosurecomprise cells in in vitro culture as well as cells that are within ahost animal.

DNA may be stably incorporated into cells or may be transientlyexpressed using methods known in the art. Stably transfected mammaliancells may be prepared by transfecting cells with an expression vectorhaving a selectable marker gene, and growing the transfected cells underconditions selective for cells expressing the marker gene. To preparetransient transfectants, mammalian cells are transfected with a reportergene to monitor transfection efficiency.

To produce such stably or transiently transfected cells, the cellsshould be transfected with a sufficient amount of the nucleic acidbinding protein-encoding nucleic acid to form the nucleic acid bindingprotein. The precise amounts of DNA encoding the nucleic acid bindingprotein may be empirically determined and optimised for a particularcell and assay.

Host cells are transfected or, preferably, transformed with theabove-captioned expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. Heterologous DNA may be introduced intohost cells by any method known in the art, such as transfection with avector encoding a heterologous DNA by the calcium phosphatecoprecipitation technique or by electroporation. Numerous methods oftransfection are known to the skilled worker in the field. Successfultransfection is generally recognised when any indication of theoperation of this vector occurs in the host cell. Transformation isachieved using standard techniques appropriate to the particular hostcells used.

Incorporation of cloned DNA into a suitable expression vector,transfection of eukaryotic cells with a plasmid vector or a combinationof plasmid vectors, each encoding one or more distinct genes or withlinear DNA, and selection of transfected cells are well known in the art(see, e.g. Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press).

Transfected or transformed cells are cultured using media and culturingmethods known in the art, preferably under conditions, whereby thenucleic acid binding protein encoded by the DNA is expressed. Thecomposition of suitable media is known to those in the art, so that theycan be readily prepared. Suitable culturing media are also commerciallyavailable.

Nucleic acid binding proteins according to the invention may be employedin a wide variety of applications, including diagnostics and as researchtools. Advantageously, they may be employed as diagnostic tools foridentifying the presence of nucleic acid molecules in a complex mixture.nucleic acid binding molecules according to the invention candifferentiate single base pair changes in target nucleic acid molecules.

Accordingly, the invention provides a method for determining thepresence of a target nucleic acid molecule, comprising the steps of:

a) preparing a nucleic acid binding protein by the method set forthabove which is specific for the target nucleic acid molecule;

b) exposing a test system comprising the target nucleic acid molecule tothe nucleic acid binding protein under conditions which promote binding,and removing any nucleic acid binding protein which remains unbound;

c) detecting the presence of the nucleic acid binding protein in thetest system.

In a preferred embodiment, the nucleic acid binding molecules of theinvention can be incorporated into an ELISA assay. For example, phagedisplaying the molecules of the invention can be used to detect thepresence of the target nucleic acid, and visualised using enzyme-linkedanti-phage antibodies.

Further improvements to the use of zinc finger phage for diagnosis canbe made, for example, by co-expressing a marker protein fused to theminor coat protein (gVIII) of bacteriophage. Since detection with ananti-phage antibody would then be obsolete, the time and cost of eachdiagnosis would be further reduced. Depending on the requirements,suitable markers for display might include the fluorescent proteins (A.B. Cubitt, et al., (1995) Trends Biochem Sci. 20, 448-455; T. T. Yang,et al., (1996) Gene 173, 19-23), or an enzyme such as alkalinephosphatase which has been previously displayed on gIII (J. McCafferty,R. H. Jackson, D. J. Chiswell, (1991) Protein Engineering 4, 955-961)Labelling different types of diagnostic phage with distinct markerswould allow multiplex screening of a single nucleic acid sample.Nevertheless, even in the absence of such refinements, the basic ELISAtechnique is reliable, fast, simple and particularly inexpensive.Moreover it requires no specialised apparatus, nor does it employhazardous reagents such as radioactive isotopes, making it amenable toroutine use in the clinic. The major advantage of the protocol is thatit obviates the requirement for gel electrophoresis, and so opens theway to automated nucleic acid diagnosis.

The invention provides nucleic acid binding proteins which can beengineered with exquisite specificity. The invention lends itself,therefore, to the design of any molecule of which specific nucleic acidbinding is required. For example, the proteins according to theinvention may be employed in the manufacture of chimeric restrictionenzymes, in which a nucleic acid cleaving domain is fused to a nucleicacid binding domain comprising a zinc finger as described herein.

Moreover, the invention provides therapeutic agents and methods oftherapy involving use of nucleic acid binding proteins as describedherein. In particular, the invention provides the use of polypeptidefusions comprising an integrase, such as a viral integrase, and anucleic acid binding protein according to the invention to targetnucleic acid sequences in vivo (Bushman, (1994) PNAS (USA)91:9233-9237). In gene therapy applications, the method may be appliedto the delivery of functional genes into defective genes, or thedelivery of nonsense nucleic acid in order to disrupt undesired nucleicacid. Alternatively, genes may be delivered to known, repetitivestretches of nucleic acid, such as centromeres, together with anactivating sequence such as an LCR. This would represent a route to thesafe and predictable incorporation of nucleic acid into the genome.

In conventional therapeutic applications, nucleic acid binding proteinsaccording to the invention may be used to specifically knock out cellhaving mutant vital proteins. For example, if cells with mutant ras aretargeted, they will be destroyed because ras is essential to cellularsurvival. Alternatively, the action of transcription factors may bemodulated, preferably reduced, by administering to the cell agents whichbind to the binding site specific for the transcription factor. Forexample, the activity of HIV tat may be reduced by binding proteinsspecific for HIV TAR.

Moreover, binding proteins according to the invention may be coupled totoxic molecules, such as nucleases, which are capable of causingirreversible nucleic acid damage and cell death. Such agents are capableof selectively destroying cells which comprise a mutation in theirendogenous nucleic acid.

Nucleic acid binding proteins and derivatives thereof as set forth abovemay also be applied to the treatment of infections and the like in theform of organism-specific antibiotic or antiviral drugs. In suchapplications, the binding proteins may be coupled to a nuclease or othernuclear toxin and targeted specifically to the nucleic acids ofmicroorganisms.

The invention likewise relates to pharmaceutical preparations whichcontain the compounds according to the invention or pharmaceuticallyacceptable salts thereof as active ingredients, and to processes fortheir preparation.

The pharmaceutical preparations according to the invention which containthe compound according to the invention or pharmaceutically acceptablesalts thereof are those for enteral, such as oral, furthermore rectal,and parenteral administration to (a) warm-blooded animal(s), thepharmacological active ingredient being present on its own or togetherwith a pharmaceutically acceptable carrier. The daily dose of the activeingredient depends on the age and the individual condition and also onthe manner of administration.

The novel pharmaceutical preparations contain, for example, from about10% to about 80%, preferably from about 20% to about 60%, of the activeingredient. Pharmaceutical preparations according to the invention forenteral or parenteral administration are, for example, those in unitdose forms, such as sugar-coated tablets, tablets, capsules orsuppositories, and furthermore ampoules. These are prepared in a mannerknown per se, for example by means of conventional mixing, granulating,sugar-coating, dissolving or lyophilising processes. Thus,pharmaceutical preparations for oral use can be obtained by combiningthe active ingredient with solid carriers, if desired granulating amixture obtained, and processing the mixture or granules, if desired ornecessary, after addition of suitable excipients to give tablets orsugar-coated tablet cores.

Suitable carriers are, in particular, fillers, such as sugars, forexample lactose, sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, furthermore binders, such as starch paste, using,for example, corn, wheat, rice or potato starch, gelatin, tragacanth,methylcellulose and/or polyvinylpyrrolidone, if desired, disintegrants,such as the abovementioned starches, furthermore carboxymethyl starch,crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof,such as sodium alginate; auxiliaries are primarily glidants,flow-regulators and lubricants, for example silicic acid, talc, stearicacid or salts thereof, such as magnesium or calcium stearate, and/orpolyethylene glycol. Sugar-coated tablet cores are provided withsuitable coatings which, if desired, are resistant to gastric juice,using, inter alia, concentrated sugar solutions which, if desired,contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycoland/or titanium dioxide, coating solutions in suitable organic solventsor solvent mixtures or, for the preparation of gastric juice-resistantcoatings, solutions of suitable cellulose preparations, such asacetylcellulose phthalate or hydroxypropylmethylcellulose phthalate.Colorants or pigments, for example to identify or to indicate differentdoses of active ingredient, may be added to the tablets or sugar-coatedtablet coatings.

Other orally utilisable pharmaceutical preparations are hard gelatincapsules, and also soft closed capsules made of gelatin and aplasticiser, such as glycerol or sorbitol. The hard gelatin capsules maycontain the active ingredient in the form of granules, for example in amixture with fillers, such as lactose, binders, such as starches, and/orlubricants, such as talc or magnesium stearate, and, if desired,stabilisers. In soft capsules, the active ingredient is preferablydissolved or suspended in suitable liquids, such as fatty oils, paraffinoil or liquid polyethylene glycols, it also being possible to addstabilisers.

Suitable rectally utilisable pharmaceutical preparations are, forexample, suppositories, which consist of a combination of the activeingredient with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols or higher alkanols. Furthermore, gelatin rectalcapsules which contain a combination of the active ingredient with abase substance may also be used. Suitable base substances are, forexample, liquid triglycerides, polyethylene glycols or paraffinhydrocarbons.

Suitable preparations for parenteral administration are primarilyaqueous solutions of an active ingredient in water-soluble form, forexample a water-soluble salt, and furthermore suspensions of the activeingredient, such as appropriate oily injection suspensions, usingsuitable lipophilic solvents or vehicles, such as fatty oils, forexample sesame oil, or synthetic fatty acid esters, for example ethyloleate or triglycerides, or aqueous injection suspensions which containviscosity-increasing substances, for example sodiumcarboxymethylcellulose, sorbitol and/or dextran, and, if necessary, alsostabilisers.

The dose of the active ingredient depends on the warm-blooded animalspecies, the age and the individual condition and on the manner ofadministration. In the normal case, an approximate daily dose of about10 mg to about 250 mg is to be estimated in the case of oraladministration for a patient weighing approximately 75 kg

The invention is described below, for the purpose of illustration only,in the following examples.

EXAMPLE 1

Determination of Binding Site Preferences in Zinc Fingers

Design of Zinc Finger Phage Display Libraries

Zinc finger-DNA recognition at the interface between adjacent DNAsubsites is studied using a zinc finger phage display library. Thislibrary is based on the three-finger DNA-binding domain of Zif268, butcontains randomisations of amino acids from finger 2 (F2) and finger 3(F3), at residue positions which could form a network of contacts acrossthe interface of their DNA subsites. The detailed design of the libraryis shown in FIG. 1 c, together with the generic DNA binding site used inselections. Briefly, the library contains randomisations at F2 residueposition 6 (hereafter denoted F2[+6]) and F3 residue positions −1, +1,+2 and +3 (hereafter denoted F3[−1], F3[+2], etc.).

Library selections are carried out using DNA binding sites thatresembled the Zif268 operator, but which contained systematiccombinations of bases in the DNA doublet which forms the base-stepbetween the DNA subsites of F2 and F3. DNA binding sites are of thegeneric form 5′-GNX-XCG-GCG-3′, where X-X denotes a given combination ofthe bases at the interface between the DNA subsites, and N denotes thatthe four bases are equally represented at DNA position 3. Thus theinteraction between F3[+3] and nucleotide position 3N is allowedcomplete freedom in this experiment. This feature of the library allowsselection of a large family (or database) of related zinc fingers thatbind a given combination of bases at nucleotide positions 4X and 5X, butwhich are non-identical owing to different interactions with the middlebase in the nominal triplet subsite of F3.

The first library to be constructed, LIB-A, contains randomisations atF2 residue position 6 and F3 residue positions −1, 1, 2 and 3 (see FIG.2), and is sorted using the DNA sequence 5′GNX-XCG-GCG-3′, where X-Xdenotes a known combination of the two bases at DNA positions 4X and 5X,and N denotes an equal probability of any of the four bases at DNAposition 3. The second library, LIB-B, contains randomisations at F2residue position 6 and F3 residue positions −1 and 2, and is sortedusing the DNA sequence 5′-GCX-XCG-GCG3′, where X-X denotes a knowncombination of the two bases at DNA positions 4X and 5X.

The genes for the two different zinc finger phage display libraries areassembled from four synthetic DNA oligonucleotides by directionalend-to-end ligation using three short complementary DNA linkers. Theoligonucleotides contain selectively randomised codons (of sequence NNS;N=A/C/G/T, S=G/C) in the appropriate amino acid positions of fingers 2and 3. The constructs are amplified by PCR using primers containing NotI and Sfi I restriction sites, digested with the above endonucleases toproduce cloning overhangs, and ligated into phage vector Fd-Tet-SN.Electrocompetent E. coli TG 1 cells are transformed with the recombinantvector and plated onto TYE medium (1.5% agar, 1% Bacto tryptone, 0.5%Bacto yeast extract, 0.8% NaCl) containing 15 μg/ml tetracycline.

Allowing this freedom to some protein-DNA interactions that are notbeing studied is a useful strategy towards increasing the diversity ofclones which can be obtained from any one selection experiment. However,at the same time, it is important to limit the number of contacts thatare allowed contextual freedom at any one time, otherwise there is adanger that a subset of particularly strong intermolecular interactionswill dominate the selections. Anticipating this eventuality, a smallersublibrary is also created that contains randomised residues only inpositions F2[+6] and F3[− and +2], and therefore does not allow forcontextual freedom in selections. Clones selected from this library aremarked with an asterisk when they are discussed herein.

Experimental Strategy

Phage selections from the two zinc finger libraries are performedseparately in order to determine the diversity of DNA sequences whichcan be bound specifically by members of each library. Sixteen selectionsare performed on each library, using the different DNA binding sitesthat correspond to all 16 possible combinations of bases at nucleotidepositions 4X and 5X. The DNA binding site used to select specificallybinding phage is immobilised on a solid surface, while a 10-fold excessof each of the other 15 DNA sites is present in solution as a specificcompetitor.

Phage Selections

Tetracycline resistant colonies are transferred from plates into 2×TYmedium (16 g/litre Bacto tryptone, 10 g/litre Bacto yeast extract, 5g/litre NaCl) containing 50 μM ZnCl₂ and 15 μg/ml tetracycline, andcultured overnight at 30° C. in a shaking incubator. Cleared culturesupernatant containing phage particles is obtained by centrifuging at300 g for 5 minutes.

Biotinylated DNA target sites (1 pmol) are bound to streptavidin-coatedtubes (Boehringer Mannheim). Phage supernatant solutions are diluted1:10 in PBS selection buffer (PBS containing 50 μM ZnCl₂, 2% Marvel, 1%Tween, 20 μg/ml sonicated salmon sperm DNA, 10 pmol/ml of each of the 15other possible unbiotinylated DNA sites), and 1 ml is applied to eachtube for 1 hour at 20° C. After this time, the tubes are emptied andwashed 20 times with PBS containing 50 μM ZnCl₂, 2% Marvel and 1% Tween.Retained phage are eluted in 0.1 ml 0.1M triethylamine and neutralisedwith an equal volume of 1M Tris (pH 7.4). Logarithmic-phase E. coli TG 1(0.5 ml) are infected with eluted phage (50 μl), and used to preparephage supernatants for subsequent rounds of selection. After 3 rounds ofselection, E. coli infected with selected phage are plated, individualcolonies are picked and used to grow phage for binding site signatureassays and DNA sequencing.

After three rounds of phage selection against a particular DNA bindingsite, individual zinc finger clones are recovered, and the DNA bindingspecificity of each clone is determined by the binding site signaturemethod. This involves screening each zinc finger phage for binding toeight different libraries of the DNA binding site, designed such thateach library contains one fixed base and one randomised base at eitherof positions 4X and 5X (i.e. libraries GN, AN, TN, CN, and NG, NA, NT,NC). Thus each of the 16 DNA binding sites used in selection experimentsis specified by a unique combination of two libraries—for example, theDNA binding site containing 4G5G is present in only two of the eightlibraries in which the relevant doublet had one nucleotide randomisedand the other nucleotide fixed as guanine, i.e. libraries 4G5N and 4N5G.The eight DNA libraries used in binding site signatures are arrayedacross a microtitre plate and zinc finger phage binding is detected byphage ELISA. The pattern of binding to the eight DNA libraries revealsthe DNA sequence specificity (or preference) of each phage clone, andonly those clones found to be relatively specific are subsequentlysequenced to reveal the identity of the amino acids present in therandomised zinc finger residue positions.

Procedures are as described previously (Choo, Y. & Klug, A. (1994) Proc.Natl. Acad. Sci. USA 91, 11163-11167; Choo, Y. & Klug, A. (1994) Proc.Natl. Acad. Sci. USA 91, 11168-11172). Briefly, 5′-biotinylatedpositionally randomised oligonucleotide libraries, containing Zif268operator variants, are synthesised by primer extension as described. DNAlibraries (0.4 pmol/well for LIB-A and 1.2 pmol/well for LIB-B) areadded to streptavidin-coated ELISA wells (Boehringer-Mannheim) in PBScontaining 50 μM ZnCl₂ (PBS/Zn). Phage solution (overnight bacterialculture supernatant diluted 1:10 in PBS/Zn containing 2% Marvel, 1%Tween and 20 μg/ml sonicated salmon sperm DNA) are appiued to each well(50 μl/well). Binding is allowed to proceed for one hour at 20° C.Unbound phage are removed by washing 6 times with PBS/Zn containing 1%Tween, then 3 times with PBS/Zn. Bound phage are detected by ELISA usinghorseradish peroxidase-conjugated anti-M13 IgG (Pharmacia Biotech) andthe colourimetric signal quantitated using SOFFMAX 2.32 (MolecularDevices).

The coding sequence of individual zinc finger clones is amplified by PCRusing external primers complementary to phage sequence. These PCRproducts are sequenced manually using Thermo Sequenase cycle sequencing(Amersham Life Science).

Analysis of Phage-Selected Zinc Fingers

FIG. 3 shows the binding site signatures of relatively sequence-specificzinc finger phages selected from both libraries, using the 16 differentDNA doublets which form the base-step between the DNA subsites offingers 2 and 3. The results show that zinc finger clones are selectedwhich bind specifically to almost all subsites, including those tripletsin which the 5′ position (nucleotide 5X in the model system) is fixed asa base other than guanine. Overall, the selections show that any of thefour bases can be bound specifically in both the 5′ and 3′ positions ofa nominal triplet subsite. The results are summarised in FIG. 4.

Selections from the smaller sub-library yield fingers that can bindspecifically to only 8 of the 16 doublets, whereas members of the largerlibrary yield fingers that recognise 15 out of the 16 doublets. It isnot known whether this difference in efficacy originates from theinclusion of more randomised positions in the larger library, or theconformational flexibility afforded by the contextual freedom designedinto the larger library, or both. The only base-step that does not yieldspecific zinc fingers is 4G5A. This dinucleotide may induce anunfavourable DNA deformation in the context of the DNA binding sitesused for selection.

EXAMPLE 2

Determination of +2 specificity for position 1

The amino acid present in α-helical position 2 of a zinc finger can helpdetermine the specificity for the base-pair at the interface of twooverlapping DNA quadruplet subsites (see FIG. 1B; position 5/5′,corresponding to position 1 or 4 of the quadruplet as discussed above).An Asp residue present in F3[+2] of wild-type Zif268 has been shown toplay a role in DNA recognition, and further examples are generated bythe current phage display experiments (See Example 1 for details, andFIG. 5A).

The experimental protocol followed is that of Example 1. FIG. 5A showsan example of related zinc finger clones showing the effect of α-helicalposition 2 on DNA-binding specificity. In this case, position 6 offinger 2 is invariant (Asn) and the change in case specificity in thezinc finger in order to select for contact to this base is dictated byposition +2 in finger 3.

This family of zinc fingers is derived from selections using DNA bindingsites containing 4T5A or 4T5C subsite interfaces. The base preferencefor the 5X-5′X base-pair is determined by the amino acid present atF3[+2], probably by the formation of cross-strand contacts.

FIG. 5B shows examples of correlations between certain amino acidsselected at F3[+2] and the identity of the base present at position 5′X.Selections reveal the possibility of DNA contacts from five amino acids(Asn, Gln, Arg, Lys and His) which are all capable of donating a H-bondto the exocyclic oxygen atom of either guanine (0₆) or thymine (0₄) innucleotide position 5′X. The clones isolated with these amino acids atF3[+2] are listed in this diagram together with the binding sitesignature showing the base-preference at position 5′X. Overall, Serdominated the selections with an occurrence of 38%, in accord with itspresence in position 2 in over half of all known zinc fingers.Threonine, Ala and Gly occurred frequently in the selections (15%, 15%and 9% respectively) but did not show any discernible patterns ofdiscrimination. Certain amino acids (Cys, Asp, Phe, Ile, Leu, Met, Pro,Val and Trp) are never selected in position 2. Their ability to bind incertain situations is however not to be excluded.

A small subset of amino acids selected in F3[+2] show significantcorrelations to the identity of the base-pair in position 5′X (FIG. 5B),suggesting that cross-strand interactions between these may be a generalmechanism of DNA-recognition. Most of these correlations can berationalised as pairings between hydrogen bond donors in F3[+2] andguanine or thymine in DNA position 5′X, in accordance with the frameworkof the Zif268 model. In contrast to amino acids that are never selectedin position 2, or amino acids that are selected but which show nosignificant correlations, the amino acids which consistently appear toplay a role in DNA recognition from this position have side chains withmultiple hydrogen bonding groups. It is possible that these residues canplay a role in base recognition because they achieve greater specificityby participating in buttressing networks.

EXAMPLE 3

Construction of a General Purpose Library

The binary library system constructed in this example compriseslibraries LIB1/2 and LIB2/3 that each encode the three fingers of Zif268but with some amino acid positions selectively randomised. Instead ofadhering to the model of modular zinc fingers, the new libraries containconcerted variations in certain amino acid positions in adjacent zincfingers. Thus LIB1/2 contains simultaneous variations in F1 positions−1, 2, 3, 5 and 6 and F2 positions −1, 1, 2 and 3. LIB2/3 containssimultaneous variations in F2 positions 3 and 6 and F3 positions −1, 1,2, 3 and 5, 6. The remaining amino acids in each library are as the WTZif268 sequence. The two libraries are cloned in Fd phage as GIIIfusions according to standard protocols.

The amino acids that are allowed at each varied position are as follows:

LIB1/2

F1 pos. −1=R, Q, H, N, D, A, T;

-   -   pos. 2=D, A, R, Q, H, K, S, N;    -   pos. 3=H, N, S, T, V, A, D;    -   pos. 5=I, T;    -   pos. 6=R, Q, V, A, E, K, N, T.

F2 pos. −1=R, Q, H, N, D, A, T;

-   -   pos. 1=S, R;    -   pos. 2=D, A, R, Q, H, K, S, N;    -   pos. 3=H, N, S, T, V, A, D;

LIB2/3

F2 pos. 3=H, N, S, T, V, A, D;

-   -   pos. 6=R, Q, V, A, E, K, N, T.    -   F3 pos. −1=R, Q, H, N, D, A, T;    -   pos. 1=R, K, S, N;    -   pos. 2=D, A, R, Q, H, K, S, N;    -   pos. 3=H, N, S, T, V, A, D;    -   pos. 5=K, 1, T;    -   pos. 6=R, Q, V, A, E, K, N, T.

Selections and Recombinations

Selections are performed using the DNA sequence GCG-GMN-OPQ for LIB 1/2and the DNA sequence IJK-LMG-GCG for LIB2/3, where the underlined basesare bound by the WT Zif268 residues and each of the other letters standsfor any given nucleotide. The conserved nucleotides of the Zif268binding site serve to fix the register of the interaction by binding tothe conserved portion of the Zif268 DNA-binding domain. The binary phagedisplay libraries can be mixed so that selections using these twogeneric sites are performed in a single tube, or the selections can beperformed separately. After a number of rounds of selection the twolibraries are recombined to produce a chimaeric DNA-binding domain thatrecognises the sequence IJK-LMN-OPQ.

The recombination reactions are performed by amplifying the selectedthree-finger domains by PCR and cutting the PCR products usingrestriction enzyme Ddel. This cuts the genes of both zinc fingerlibraries at the DNA sequence coding for F2 α-helical positions 4 and 5.The digested products are randomly religated to produce recombinantgenes coding for the chimaeric DNA-binding domains (and other productsincluding reconstituted WT Zif268). The chimaeric DNA-binding domainsare selectively amplified from the mixture of products by PCR usingselective primers that recognise the recombinant F1 and F3 genes, ratherthan WT genes, and cloned in Fd phage (for more selections) or othervectors (e.g. for expression in E. coli).

The initial selections from the binary libraries can be pushed tocompletion, thus allowing the assembly of a single clone byrecombination. Alternatively, if the initial selections are lessstringent, many candidates will be available for the assembly of variouschimaeric domains after recombination. In the latter case, the bestrecombinant protein can be selected by further rounds of selection onphage.

1. A method of providing a zinc finger protein comprising first, secondand third zinc fingers, each having positions −1 to 9 with position 1representing the first amino acid of an alpha-helix, the methodcomprising: selecting a first zinc finger protein comprising first andsecond zinc fingers from a first library of zinc finger proteins atleast partially randomized in at least positions 6 and 2 of the firstand second fingers respectively; selecting a second zinc finger proteincomprising second and third zinc fingers from a second library of zincfinger proteins at least partially randomized in at least positions 6and 2 of the second and third fingers respectively; forming a third zincfinger protein comprising first, second and third zinc fingers byrecombination in the second zinc fingers of the first and second zincfinger proteins.
 2. The method of claim 1, wherein the first library ofzinc finger proteins is at least partially randomized at positionsselected from the group consisting of positions −1, 1, 2, 3, 5 and 6 inthe first zinc finger and at positions selected from the groupconsisting of positions −1 1, 2 and 3 in the second zinc finger.
 3. Themethod of claim 1, wherein the second library of zinc finger proteins isat least partially randomized at positions selected from the groupconsisting of positions 3, 5, and 6 in the second finger and atpositions selected from the group consisting of positions −1, 1, 2, 3, 5and 6 in the third finger.
 4. The method of claim 3, wherein the secondlibrary of zinc finger proteins is at least partially randomized atpositions selected from the group consisting of positions 3, 5 and 6 inthe second finger and at positions selected from the group consisting ofpositions −1, 1, 2, and 3 in the third finger.
 5. The method of claim 1,wherein the recombination occurs between positions 3 and 5 of the secondzinc fingers of each of the first and second zinc finger proteins. 6.The method of claim 5, wherein the recombination occurs betweenpositions 4 and 5 of the second zinc fingers of the first and secondzinc finger proteins.
 7. The method of claim 1, wherein the third zincfinger protein is formed by expressing a nucleic acid encoding the thirdzinc finger protein.
 8. The method of claim 1, wherein the randomizationof amino acid residues is restricted such that the following amino acidsappear at the given positions: Position Amino Acids −1 R, Q, H, N, D, A,T 1 S, R, K, N 2 D, A, R, Q, H, K, S, N 3 H, N, S, T, V, A, D 5 I, T, K6 R, Q, V, A, E K, N, T.